Intramuscular inoculation of Sin Nombre hantavirus cDNAs induces cellular and humoral immune responses in BALB/c mice.

To examine whether genetic immunization with Sin Nombre (SN) hantavirus genes could elicit immune responses, nine fragments spanning the envelope glycoprotein genes G1 and G2, and the complete N gene were cloned into a CMV expression vector. To ensure representation of all potential epitopes, adjacent fragments of the glycoprotein genes overlapped one another by 100 nucleotides. Vectors containing the gene fragments were inoculated intramuscularly into BALB/c mice and splenocyte proliferation and western blot-detectable antibodies and neutralization titers were determined. The N gene and seven of the nine M segment-derived cDNAs tested produced significant specific lymphoproliferative responses, and many of the constructs elicited either neutralizing or western blot-detectable antibodies. These promising results encourage the development of infection models for SN virus that will be capable of detecting protective responses.

The effect of fluticasone propionate on respiratory syncytial virus-induced chemokine release by a human bronchial epithelial cell line.

Respiratory syncytial virus (RSV) is an important cause of bronchiolitis in infants, is an important trigger of asthma exacerbation, and stimulates chemokine production by human respiratory epithelial cells in vitro. We tested the effect of the corticosteroid fluticasone propionate (FP) on RSV-stimulated production of the chemokines interleukin 8 (IL-8) and RANTES (regulated upon activation, normal T cell expressed and secreted) by a human bronchial epithelial cell line, BEAS-2B. Confluent BEAS-2B cultures were inoculated with RSV at approximately 1 plaque-forming unit/cell, and media were collected at 24 h intervals. Concentrations of IL-8 and RANTES were measured in supernatants using ELISA. The effect of FP at varying concentrations on RSV-induced chemokine release was determined. RSV stimulated increased release of both IL-8 and RANTES, particularly at 24-48 h after virus inoculation. Significant but incomplete inhibition of RSV-stimulated increases for both chemokines was found when cultures were treated with FP at > or = 10(-8) M (for IL-8) or > or = 10(-7) M (for RANTES). There was no significant effect of FP on release of RSV itself from infected BEAS-2B cells. We conclude that a possible mechanism for the efficacy of inhaled corticosteroids in reducing the frequency or severity of asthma exacerbations is inhibition of virus-induced chemokine production by airway cells.

Ferritin expression after in vitro exposures of human alveolar macrophages to silica is iron-dependent.

The increased availability of catalytically active iron after silica exposure can present an oxidative injury to a living system. Sequestration of reactive iron would, therefore, confer a protective effect. The intracellular storage of iron by ferritin within macrophages can limit the potential for radical generation and cellular injury resulting from exposure to a metal chelate. We tested the hypothesis that in vitro exposure of human alveolar macrophages to silica increases the expression of ferritin through a posttranscriptional mechanism. Exposure of 1.0 x 10(6) macrophages to 100 microg/ml silica for 4 h increased light-subunit (L)-ferritin protein concentrations in both cell supernatants and lysates. Inclusion of 1.0 mM deferoxamine in the reaction mixtures inhibited increases in ferritin after silica. To test for a posttranscriptional regulation of ferritin protein expression, cells were incubated with acid-washed particles, silica with complexed zinc cation, and silica with complexed iron cation. L-ferritin protein concentrations were increased in both cell supernatants and lysates after 4 h of exposure to silica with complexed iron cation. There were no increases in L-ferritin after incubations with acid-washed particles or silica with complexed zinc cation. There were no significant differences in levels of L-ferritin cDNA between any of the exposures, suggesting a posttranscriptional control of ferritin expression.

Cytokine production by cultured human bronchial epithelial cells infected with a replication-deficient adenoviral gene transfer vector or wild-type adenovirus type 5.

Exposure of animals to adenoviral gene transfer vectors has been associated with respiratory tract inflammation. The pathogenesis of this inflammation is unclear. One hypothesis is that viral vectors directly induce production of inflammatory cytokines by host cells in the airways. We exposed cultured human lung cells to an adenovirus-5--based vector containing the cytomegalovirus promoter and lacZ reporter gene (Ad.CMV.lacZ) and to wild-type adenovirus 5 (wtAd5) and measured subsequent release of cytokines into cell culture supernatants. Inoculation of human bronchial epithelial (HBE) cells with Ad.CMV. lacZ at 10(1) to 10(4) plaque-forming units (pfu)/cell resulted in dose-related expression of lacZ by both X-gal staining and immunohistochemistry but did not increase release of interleukin (IL)-8 or IL-6 at 24, 48, or 96 h after inoculation. In the same cultures, tumor necrosis factor-alpha induced marked increases in release of both IL-8 and IL-6 at 24 and 48 h after stimulation. Similar data were observed in the BEAS-2B HBE cell line. HBE cells incubated with wtAd5 at doses of 10(1) to 10(3) pfu/cell did not release increased amounts of IL-6 or IL-8 up to 48 h after inoculation, though wild-type respiratory syncytial virus (3 pfu/HBE cell) infection resulted in increases in both cytokines. Human alveolar macrophages obtained by bronchoalveolar lavage also showed no increases in cytokine release after incubation with Ad.CMV.lacZ, though relatively little gene transfer occurred in macrophages. These data do not support a role for direct induction of airway epithelial or alveolar macrophage inflammatory cytokines in the pathogenesis of inflammation associated with exposure of airways to adenovirus or to adenoviral gene transfer vectors.

Nasal cytokine production in viral acute upper respiratory infection of childhood.

Children in a day care center underwent serial nasal lavages in order to assess nasal cytokine expression during acute upper respiratory infections (URI). Interleukin (IL)-1 beta, IL-8, IL-6, and tumor necrosis factor-alpha (TNF-alpha) were markedly elevated in nasal lavage fluid during acute URI compared to baseline, and all except TNF-alpha decreased significantly by 2-4 weeks later. Cytokine patterns in respiratory syncytial virus-positive and -negative illnesses did not differ significantly. A subgroup of children also underwent superficial mucosal biopsy under the inferior nasal turbinate. During acute URI, biopsy cells (90%-95% epithelial) showed increased transcripts for IL-1 beta, IL-8, and IL-6 in 7 of 9 subjects, suggesting that epithelial cells may be one source of cytokines during acute URI. The results show that inflammatory cytokines are elevated in nasal secretions during acute URI in preschool children. Thus, cytokines are likely to participate in regulation of respiratory virus-induced inflammation.

The importance of the 3'-untranslated region in the translational control of ferritin mRNA.

Ferritin synthesis provides a dramatic example of translational control; stored ferritin mRNA is translated at relatively low rates which can increase 40-50 times when cellular iron levels increase. Although it is not known if agents other than cellular iron levels can release the repression of ferritin mRNA in vivo, the repression appears to be eliminated during the isolation of poly(A+) RNA, judged by translation in wheat germ lysates (WG). Using the bullfrog tadpole as a model, because of the abundance of ferritin-rich embryonic red cells, we now show specific repression of ferritin mRNA in the isolated poly(A+) RNA translated in rabbit reticulocyte lysates (RR) (RR/WG = 25%). Repression of ferritin mRNA was associated with the inability to form polyribosomes in analogy to iron-poor cells in vivo. The addition of various complexes of iron did not relieve the repression, suggesting that in vivo at least part of the effect of iron may be indirect and mediated by factors absent in the cell-free system; all three ferritin subunit mRNAs (H, M, and L) appeared to be regulated coordinately in vitro and in vivo as well. Comparison of transcripts of DNA encoding the M subunit of ferritin, but containing deletions in the 3'-untranslated (UT) region, showed that a region 70 nucleotides long was important for repression. Comparison of secondary structures predicted for the eight known ferritin subunit mRNAs from humans, rats, chickens, and frogs indicates that a region involved in base pairing common to all the mRNAs is eliminated when the 3'-UT region is shortened to 24 nucleotides. Although regions in the 5'-UT of mRNAs, including ferritin, have been shown to be involved in translational regulation, it is clear that complete regulation can involve both the 3'-UT and the 5'-UT regions, mediated, presumably, by secondary and tertiary interactions along the mRNA molecule.